NATURE REVIEWS | GENETICS VOLUME 2 | APRIL 2001 | 241

Y is the answer

S E X D E T E R M I N AT I O N

Few tasks are sex-specific these days,but biology makes some exceptions.Possibly the most fundamental exam-ple of these is the molecular route bywhich sex is determined. In flies, thisinvolves two crucial events: the deci-sion of which of the two sexual devel-opment pathways to follow, and theadjustment of X-chromosomeexpression to the level appropriate tothat sex. Critical genes are those thatlie upstream of these two processes,and can be identified by searching formutations the lethal effects of whichdepend on the X-chromosome dose(known as sex-specific lethals). Thegene flex (female-specific lethal on X)was thought to be among these, but arecent study questions previous con-clusions and shows that the femalelethality caused by flex depends onthe absence of the Y chromosome,rather than on the presence of two Xchromosomes.

The Drosophila gene Sxl (Sexlethal) lies at the heart of sex determi-nation — Sxl protein is activated (bythe double X dosage) only in females,in which an autoregulatory loopinvolving pre-mRNA splicing ensurescontinued expression of the female-specific protein isoforms. In males,which are XY, no active Sxl exists. Sxlis crucial for female development anddosage compensation; moreover, thepresence of Sxl in males is lethal. Anearlier study had proposed flex to be apositive regulator of Sxl on the basisof, among other things, the ability ofa flex mutant to rescue the male

lethality of the SxlM gain-of-functionalleles, which cause the female iso-form of Sxl to be expressed in males.In his study, Tom Cline witnessed nophenotypic suppression by flex ofSxlM alleles — not even weak ones —and so sought to find a more con-vincing explanation for the flex phe-notype itself. This came from anoften neglected source: the Y chro-mosome. This chromosome rescuesthe flex-induced female lethality,whereas flex males that lack the Ychromosome (that is, are XO) do notsurvive.

Which gene(s) on the Y chromo-some could be responsible for suchan effect? The most likely candidate isthe bb (bobbed ) locus (which encodesribosomal RNAs (rRNA)) — the onlyfly locus that has alleles on both the Xand Y chromosomes. Indeed, the lackof complementation between flex anda bb− Y chromosome, together withthe revised map position of flex,might just prove the allelism betweenflex and bb.

There are two possible conclu-sions to this story — either flex hasnothing to do with Sxl (or sex deter-mination) and is yet another rRNAlocus; or this rRNA molecule has animportant function early in sex deter-mination. Either way, this work alertsresearchers that the Y chromosome isignored at their peril.

Tanita Casci

References and linksORIGINAL RESEARCH PAPER Cline, T. W. A lesson from flex: consider the Y chromosomewhen assessing Drosophila sex-specific lethals.Development 128, 1015–1018 (2001)FURTHER READING Bhattacharya, A. et al. flex,an X-linked female-lethal mutation in Drosophilamelanogaster controls the expression of Sex-lethal. Development 126, 5485–5493 (1999) WEB SITE Tom Cline’s lab

Fly images courtesy of Tom Cline, University of California, Berkeley, USA.

Fishing for new tools

T E C H N O LO G Y

One gadget noticeably absent from the zebrafish geneticist’stoolkit is an embryonic cell line to use for gene-targetingexperiments. But the wait for this vital tool might now be over, with the publication of a technique that maintainsundifferentiated, zebrafish embryonic cells in culture, which— when transplanted into embryos — can contribute to thezebrafish germ line.

The secret to the authors’ success was their use of a rainbow trout spleen cell line (called RTS34st) to create amonolayer of feeder cells on which they cultured cells derivedfrom gastrula-stage zebrafish embryos. When these embryocells are cultured without feeder cells, they cease to express the primordial germ-cell marker vasa and begin todifferentiate after five days. By contrast, when cultured on this feeder layer, embryo cells remain undifferentiated andexpress vasa for at least 25 days. Embryo cells cultured withoutfeeder cells but with RTS34st-conditioned medium alsoexpress vasa but show earlier signs of differentiation.

But can these cultured cells contribute to the zebrafishgerm line — something previous embryonic cell lines havefailed to do? To test this, Ma et al. injected cultured embryocells derived from a pigmented, neo-expressing transgeniczebrafish strain into unpigmented (GASSI) zebrafish embryos.Of the embryos that survived injection, four producedpigmented, neo-positive offspring when crossed to GASSImates (see picture), indicating that they were germlinechimeras. Although the frequency and the degree of germlinechimerism are low among the fish that survived injection,the authors reason that these potential barriers to success are offset by the large numbers of offspring that fish produce— this provides many embryos for injection and means that fish with only low rates of chimerism can still producetransgenic offspring.

The next step for Ma et al. is to establish longer-termcultures to allow time for gene targeting and selection. Thismight require further investigation into what the feeder cellsprovide to embryo cells to keep them undifferentiated.

Jane Alfred

References and linksORIGINAL RESEARCH PAPER Ma, C., Fan, L., Ganassin, R., Bols, N. & Collodi, P.Production of zebrafish germ-line chimeras from embryo cell cultures. Proc. Natl Acad. Sci.USA 98, 2461–2466.WEB SITE Paul Collodi’s lab

Unpigmented, chimeric zebrafish (top) and its pigmented transgenic offpsring. Courtesy ofPaul Collodi, Purdue University, Indiana, USA.

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